Culture medium for detecting and/or discriminating enterococcus and method therefor

ABSTRACT

The invention concerns a culture medium for isolating  enterococcus  comprising violet crystal, and preferably gram-negative bacteria inhibitors and chromogens. The invention also concerns a method for detecting  enterococcus  using said medium.

The present invention relates to a chromogenic culture medium intendedfor identifying enterococci.

In the clinical field, the detection of enterococci is very importantbecause of the appearance of strains which are resistant to antibiotics,in particular to vancomycin, the antibiotic most widely used fortreating infections. These nosocomial infections can endanger the livesof the patients affected.

Streptococci isolated from the intestine or group D streptococci wereconventionally distinguished as two groups according to theirphysiological characteristics:

-   -   “enterococci” (Streptococcus faecium and Streptococcus faecalis)        capable of growing under hostile conditions;    -   “non enterococci group D streptococci” (Streptococcus bovis and        Streptococcus equinus) incapable of multiplying under hostile        conditions.

In 1982 and 1983, genomic studies (DNA—rRNA hybridizations, theestablishment of dictionaries of oligonucleotides of 16S rRNA) showedthat “enterococci” and “non enterococcal group D streptococci” belongedto two different genera.

In 1984, Schleifer and Kilpper-Bälz, while studying the results ofDNA-23S rRNA hybridizations and DNA-DNA hybridizations confirmed theearlier results and these two authors proposed transferring the“enterococcal group of streptococci” to the genus enterococcus which isdistinct from the genus streptococcus (Int. J. Syst. Bacteriol., 1984,34, 31-34).

A very good study on enterococci can be found at the sitewww.bacterio.cict.fr/bacdico/ee/enterococcus.html, or at the sitewww.life.umd.edu/classroom/bsci424/PathogenDescriptions/Enterococcus.htm,or at the site www.enterococcus.ouhsc.edu/lab methods.asp.

It is therefore important to have a reliable and quick test which makesit possible to detect contaminations by these bacteria, which testshould be both sensitive and specific.

Enterococci generally grow in temperature ranges from 10° to 45° C., theoptimum growth being around 35° C., on non-selective medium (blood agaror chocolate agar). However, such a medium also allows the growth ofother bacteria and does not make it possible to effectively distinguishenterococci.

There are now certain culture media for the detection of enterococci, inparticular the MacConkey Agar medium, without Crystal Violet, sold byDifco. The Crystal Violet is a compound which inhibits the growth ofGram-positive bacteria, which is considered to inhibit the growth ofenterococci and of staphylococci (it should be recalled that enterococciare Gram-positive cocci which appear in isolation or in pairs or inshort chains). The “Difco Manual”, 11th edition, page 288, indicates“MacConkey Agar w/o CV (Crystal Violet) is a differential medium that isless selective than MacConkey Agar. The lack of crystal violet permitsthe growth of Staphylococcus and Enterococcus”.

This medium also contains other growth inhibitors such as bile salts.These other inhibitors are effective for inhibiting the growth ofGram-positive bacteria, but not enterococci.

Indeed, enterococcal bacteria can also grow in media containing bilesalts, the colonies not dissolving after exposure to bile. It is alsopossible to grow enterococcal bacteria in a medium containing an NaClconcentration of 6.5%, the streptococci not possessing this property.

From these observations that media for enterococci do not containCrystal Violet, but contain other inhibitors instead, it can beconcluded that, under numerous conditions, Crystal Violet is aninhibitor of enterococcal growth. This hypothesis is reinforced by thefact that among the various MacConkey media, the one used to detectenterococci does not contain Crystal Violet, but contains bile salts.However, this medium also allows the growth of staphylococci.

It should be noted that the method most commonly used for the detectionof enterococci consists of a filtration technique on a selective medium(such as the m-Enterococcus agar medium=Slanetz and Bartley medium oroxolinic acid-esculin-azide medium=OAA medium) followed by confirmationby culture on bile-esculin agar incubated for 48 hours at 44° C. Theselective effect may be reinforced by incubating the Slanetz and theBartley medium at 41° C. Most often, complete identification is notobtained and only the identification of catalase is carried out in orderto eliminate certain staphylococcal strains which are capable ofdeveloping on the media used.

It is therefore advisable to have a medium allowing the detection ofenterococci and which also makes it possible to differentiate them fromstaphylococci, which grow in general on culture media for enterococci.

In the context of the present invention, it has been shown that it ispossible to adjust the concentration of Crystal Violet such that itretains its role of growth inhibition for most Gram-positive bacteria,and in particular staphylococci, while allowing the growth ofenterococci.

Thus, the prior art media (in particular the MacConkey Agar medium soldby Difco Laboratories) generally use a Crystal Violet concentrationequal to 1 mg/l. In the context of the present invention, it has beenshown that the addition of Crystal Violet at a concentration of 0.1 upto about 1.5 mg/l makes it possible to maintain the growth ofenterococci, in particular when the medium does not contain other growthinhibitors for Gram-positive bacteria.

It has also been shown that the addition of Crystal Violet makes itpossible to avoid using sodium azide which is very often present inenterococci detection media (for its properties of inhibiting the growthof certain bacteria, but which is toxic for humans). Preferably, amedium according to the invention will not therefore contain sodiumazide.

This result, and in particular the fact that the Crystal Violetconcentration may be greater than 1 mg/l under certain conditions whileallowing the growth of enterococci, could not have been predicted in thelight of the knowledge described in the prior art, in particular thefact that it is also possible to eliminate sodium azide.

Thus, the subject of the present invention is a culture medium fordetecting and/or distinguishing enterococci, characterized in that itcontains, in a culture medium for enterococci, Crystal Violet at aconcentration allowing the growth of enterococci and the inhibition ofthe growth of most Gram-positive bacteria.

Preferably, said concentration is greater than 0.1 mg/l, most preferablygreater than 0.25 mg/l, or greater than 0.5 mg/l. Thus, inhibition ofthe growth of a large quantity of Gram-positive bacteria is observedafter addition of Crystal Violet at a concentration as low as 0.1 mg/l.

In a particular embodiment of the invention, said concentration is lessthan 1.5 mg/l, more preferably less than 1 mg/l. When the Crystal Violetconcentration is about 1 mg/l or greater than this value, it is thenadvisable to reduce, or even eliminate the other inhibitors ofGram-positive bacteria, in particular the bile salts. It is within thecapability of persons skilled in the art to adjust the concentrations ofinhibitors according to the concentration of Crystal Violet added to themedium according to the invention. In another embodiment of theinvention, the Crystal Violet concentration is less than 0.8 mg/l, morepreferably less than 0.7 mg/l. At such concentrations, it is possible tooptionally add other inhibitors of Gram-positive bacteria.

A person skilled in the art knows what the term “culture medium forenterococci” means, that is to say a culture medium containing thenutrients necessary to allow the growth of these bacteria. There may bementioned in particular peptone from casein, from soybean, from meat,from yeast extract or from beef, dextrose and the like.

Preferably, the medium according to the invention further comprises atleast one chromogenic agent, a substrate for an enzyme for fermentingsugars, said enzyme being preferably a glucosidase, in particularβ-glucosidase or a galactosidase, in particular β-galactosidase.

The fact that it is possible to add a chromogenic agent is completelyunexpected since Crystal Violet already colors the media according tothe invention, which therefore dissuades from adding a constituentproviding color, such as a chromogenic agent.

Preferably, said chromogenic agent releases, by hydrolysis, aprecipitable chromophore chosen from indoxyl, haloindoxyl (bromoindoxyl,chloroindoxyl, fluoroindoxyl, iodoindoxyl, dichloroindoxyl,chlorobromoindoxyl, trichloroindoxyl), methylindoxyl or hydroxyquinolinederivatives, in particular the following derivatives: 6-chloroindoxyl,5-bromoindoxyl, 3-bromoindoxyl, 6-fluoroindoxyl, 5-iodoindoxyl,4,6-dichloroindoxyl, 6-7-dichloroindoxyl, 5-bromo-4-chloroindoxyl,5-bromo-6-chloroindoxyl, 4,6,7-trichloroindoxyl, N-methylindoxyl or8-hydroxyquinoline.

Preferably, said chromogenic substrate for β-glucosidase is anindoxylglucoside, in particular 5-bromo-4-chloro-3-indoxyl-β-glucosideand/or said chromogenic agent, a substrate for β-galactosidase, is anindoxylgalactoside, in particular5-bromo-6-chloro-3-indoxyl-β-galactoside.

In order to better allow the detection of enterococci, it is alsopossible to add, to the culture medium according to the invention,growth inhibitors for Gram-negative bacteria, such as nalidixic acid orcolistin.

To detect atypical enterococci which are resistant to certainantibiotics, and which are largely responsible for nosocomialinfections, it is also possible to add said antibiotics to the mediaaccording to the invention. Vancomycin will thus be added at aconcentration of about 6 mg/l. It should be noted that it is possible todetect the proportion of resistant bacteria in a sample by plating on adish without antibiotics and a dish containing them and by comparing thenumber of colonies identified as enterococci on each dish.

The invention also relates to the use of a culture medium according tothe invention for detecting and/or distinguishing enterococci, and to amethod for detecting and/or distinguishing enterococci in a sample,characterized in that it comprises the steps consisting in:

-   -   a. inoculating a culture medium according to the invention with        said sample of an inoculum derived from the sample,    -   b. detecting the presence of enterococci on said culture medium.

The presence of enterococci is detected by the growth of the colonies onthe medium, and this is helped by their coloration after releasing thechromophore from the substrate chromogen for the enzyme.

A chromophore would be preferably chosen which has a wavelengthdifferent from the wavelength of Crystal Violet so as to identify itmore easily. However, the medium according to the invention also allowsthe use and the detection of chromophores having a wavelength close tothe wavelength of Crystal Violet.

In general, the invention also relates to a culture medium for thedetection of Gram-positive or Gram-negative bacteria comprising, inaddition to growth factors for said Gram-positive or Gram-negativebacteria, a chromogenic agent and Crystal Violet, the Crystal Violetbeing present at a concentration allowing the growth of said bacteriawhich it is sought to detect and the differential inhibition of thegrowth of Gram-positive bacteria, said concentration being preferablybetween 0.1 and 1.5 mg/l.

The invention also relates to the use of Crystal Violet as agrowth-selective inhibitor for the preparation of a culture medium forthe detection of Gram-positive or Gram-negative bacteria, additionallycontaining a chromogenic agent.

The invention has therefore demonstrated that it is possible to add acolorant to a chromogenic medium, while retaining the chromogenicproperties.

The chromogen is chosen such that the chromophore released has awavelength different from that of Crystal Violet or the colorant used inthe chromogenic medium.

EXAMPLES Example 1

A preferred medium for carrying out the invention comprises (for oneliter): Agar  15 g Yeast extract and peptones   9 g NaCl   5 g Nalidixicacid  50 mg Colistin   5 mg Crystal Violet 0.5 mg5-bromo-4-chloro-3-indoxyl-β-glucoside  50 mg

Example 2

Plating of bacteria on the medium according to the invention gave thefollowing results (24 hours of incubation at 37° C.). Growth ColorEnterococci + Mauve-blue Staphylococci − —

The use of the medium according to the invention therefore makes itpossible to detect the enterococci, and to distinguish them fromstaphylococci.

Example 3

Examples of media for enterococci which may be used in the context ofthe present invention, by adding Crystal Violet thereto and byoptionally removing sodium azide therefrom, or by adding chromogenicagents, and optionally vancomycin, for detecting resistant strains.

Bile-esculin medium (composition in grams by liter): Meat extract: 3.0Meat peptone: 5.0 Beef bile: 40.0 Esculin: 1.0 Iron citrate: 0.5 Agar:14.5

This medium may be enriched with 5% horse serum.

Bile-esculin-azide medium (composition in grams by liter): Tryptone:17.0 Peptone: 3.0 Yeast extract: 5.0 Esculin: 1.0 NaCl: 5.0 Ammoniacaliron citrate: 0.5 Sodium citrate: 1.0 Sodium azide: 0.25 Beef bile: 10.0Agar: 13.5

Slanetz and Bartley medium or m-Enterococcus agar (composition in gramsby liter): Tryptone: 15.0 Peptone: 5.0 Yeast extract: 0.5 Glucose: 2.0or 5.0 K₂HPO₄: 4.0 Sodium azide: 0.4 2,3,5-triphenyltetrazolium chloride10.0 (solution at 1 percent): Agar: 10

Oxolinic acid-esculin-azide medium or OAA medium (composition in gramsper liter): Tryptone: 20.0 Yeast extract: 5.0 Glucose: 1.0 NaCl: 5.0Ammoniacal iron citrate: 0.5 Sodium citrate: 1.0 Sodium azide: 0.4Oxolinic acid: 0.005 Agar: 10.0

Esculin-azide-kanamycin agar (composition in grams per liter) Tryptone:20.0 Yeast extract: 5.0 Glucose: 1.0 NaCl: 5.0 Ammoniacal iron citrate:0.5 Sodium citrate: 1.0 Sodium azide: 0.15 Kanamycin sulfate: 0.02 Agar:10.0

1. A culture medium for detecting and/or distinguishing enterococci,characterized in that it contains, in a culture medium for enterococci,Crystal Violet at a concentration allowing the growth of enterococci andthe inhibition of the growth of most Gram-positive bacteria, saidconcentration being between 0.1 and 1.5 mg/l.
 2. The culture medium asclaimed in claim 1, characterized in that it additionally comprises atleast one chromogenic agent, a substrate for an enzyme for sugarfermentation.
 3. The medium as claimed in claim 2, characterized in thatsaid enzyme is a glucosidase, in particular β-glucosidase or agalactosidase, in particular β-galactosidase.
 4. The culture medium asclaimed in claim 2 or 3, characterized in that said chromogenic agentreleases, by hydrolysis, a precipitable chromophore chosen from indoxyl,haloindoxyl (bromoindoxyl, chloroindoxyl, fluoroindoxyl, iodoindoxyl,dichloroindoxyl, chlorobromoindoxyl, trichloroindoxyl), methylindoxyl orhydroxyquinoline derivatives, in particular the following derivatives:6-chloroindoxyl, 5-bromoindoxyl, 3-bromoindoxyl, 6-fluoroindoxyl,5-iodoindoxyl, 4,6-dichloroindoxyl, 6-7-dichloroindoxyl,5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl,4,6,7-trichloroindoxyl, N-methylindoxyl or 8-hydroxyquinoline.
 5. Theculture medium as claimed in claim 3 or 4, characterized in that saidβ-glucosidase substrate is an indoxylglucoside, and/or saidβ-galactosidase substrate is an indoxyl-galactoside.
 6. The culturemedium as claimed in claim 5, characterized in that the β-glucosidasesubstrate is 5-bromo-4-chloro-3-indoxyl-β-glucoside and/or theβ-galactosidase substrate is 5-bromo-6-chloro-3-indoxyl-β-galactoside.7. The culture medium as claimed in one of claims 1 to 6, characterizedin that it also contains growth inhibitors for Gram-negative bacteria.8. The culture medium as claimed in one of claims 1 to 7, characterizedin that it comprises (for one liter): Agar  15 g Yeast extract andpeptones   9 g NaCl   5 g Nalidixic acid  50 mg Colistin   5 mg CrystalViolet 0.5 mg 5-bromo-4-chloro-3-indoxyl-β-glucoside  50 mg


9. The culture medium as claimed in one of claims 1 to 8, additionallycontaining antibiotics.
 10. The culture medium as claimed in one ofclaims 1 to 9, characterized in that it does not contain sodium azide.11. The use of a culture medium as defined in one of claims 1 to 10 fordetecting and/or distinguishing enterococci.
 12. A method for detectingand/or distinguishing enterococci in a sample, characterized in that itcomprises the steps consisting in: a. inoculating a culture medium asdefined in one of claims 1 to 10 with said sample of an inoculum derivedfrom the sample, b. detecting the presence of enterococci on saidculture medium.
 13. A culture medium for the detection of Gram-positiveor Gram-negative bacteria comprising, in addition to growth factors forsaid Gram-positive or Gram-negative bacteria, a chromogenic agent andCrystal Violet, the Crystal Violet being present at a concentrationallowing the growth of said bacteria which it is sought to detect andthe differential inhibition of the growth of Gram-positive bacteria,said concentration being between 0.1 and 1.5 mg/l.
 14. The use ofCrystal Violet as a growth-selective inhibitor for the preparation of aculture medium for the detection of Gram-positive or Gram-negativebacteria, additionally containing a chromogenic agent.